Use of organ-culture, irradiation and adoptive-transfer to investigate the role of the Xenopus thymus in T lymphocyte development
This Thesis attempts to develop an amphibian model system for exploring the role of the thymus, particularly its stromal cells, in the acquisition of allotolerance. Inbred, clonal and cytogenetically-marked Xenopus are used in this work. The initial experiments (Chapter 2) describe in vitro attempts to deplete the larval and post-metamorphic thymus of its lymphocyte component, while leaving its stromal elements intact. The histologic effects of deoxyguanosine-treatment and 6 -irradiation on the organ-cultured thymus are examined in wax sections. In vivo whole-body irradiation (3000R) and subsequent in situ residence for ~ 10 days proves the most successful technique for depleting thymic lymphocyte numbers from the froglet thymus (Chapter 3). This technique provides a lymphocyte-depleted thymus with a fairly normal 3-d stromal network. Furthermore, these thymuses show no sign of lymphocyte regeneration when organ-cultured for ~ 2 weeks. In Chapter 3, 1 um sections and electron microscopy of plastic-embedded sections provides a detailed picture of the froglet thymus following irradiation. Chapters 4 and 5 employ ploidy labelling and the X. borealis (quinacrine-fluorescence) cell marker system, to show that larval and adult (normal or in vivo irradiated) thymuses, implanted to early- thymectomized Xenopus (MHC-compatible. or -incompatible), become infiltrated by host lymphoid cells, the thymic epithelium remaining donor-derived. A time-course study shows that for normal thymus implants, host cells begin to immigrate in good number only after metamorphosis is complete; with these thymus implants, donor-derived lymphocytes can still be found in the blood and spleen of thymectomized hosts several months post-implantation. Irradiated thymus implants attracted host cells more rapidly, their lymphoid complement becoming almost exclusively host-type within 2 weeks post-implantation when animals were at a late larval stage of development. Despite rapid colonization of irradiated implants by host lymphoid cells, these thymuses degenerate soon after metamorphosis, presumably due to irradiation damage of stromal elements. The experiments in Chapter 6 compare the proliferative responses of thymocytes from normal and organ-cultured thymuses. The technical conditions (e.g. cell numbers per well) for obtaining good stimulation indices with T cell mitogens, and in mixed leucocyte culture (MLC), are examined. Thymocytes organ-cultured for 12 days generally display elevated 3HTdR uptake compared with cells tested straight from the animal. Both control and experimental (mitogen- or alloantigen-treated) cultures of organ-cultured thymocytes show these elevated DPM. Surprisingly, in vivo-irradiated, organ-cultured thymocytes, are still stimulated by phytohaemagglutinin (PHA)-treatment. Chapter 7 investigates in vitro T-cell proliferative responses of splenocytes and thymocytes in allothymus-implanted, early- thymectomized Xenopus. Splenocyte reactivity to PHA and to third- party alloantigens (in MLC) is restored when normal allogeneic thymus is implanted ("adoptively-transferred"). However, MLC reactivity of thymocytes and splenocytes to thymus-donor strain cells is generally lacking; allothymus-implanted animals are also tolerant to thymus-donor strain skin grafts. Unfortunately, thymectomized animals implanted with in vivo irradiated, allothymuses died prior to in vitro assaying. Preliminary attempts to generate supernatants (by treating cultured splenocytes with PHA and/or phorbol myristate acetate) that would routinely enhance T cell proliferative responses of thymocytes, are outlined in Chapter 8. General conclusions to be drawn from this Thesis and suggestions for future work with this amphibian model are to be found in Chapter 9.